A significant gap in understanding the structure and molecular composition of the kinetochore outer plate limits a detailed dissection of the underlying functional mechanisms. The long-term goal of this research is to dissect the molecular organization of the outer plate in vertebrate kinetochores with sufficient detail in order to determine its major functional mechanisms. The objective here is to create an accurate structural model of the outer plate that specifies spatial arrangements of selected molecular components and explains the role of these components in forming the outer plate and its attachments to microtubules. The central hypothesis is that the kinetochore outer plate consists of a fibrous network with AF15q14, CENP-F, and the Heel complex involved in forming both the network and end-on MT attachments, the Mis12 complex primarily involved in attaching the network to inner centromere, Cep57 primarily involved in MT attachments, and Zwint primarily involved in corona formation. The rationale for this project is that the new model it produces will provide a foundation for understanding how the kinetochore outer plate attaches microtubule plus ends and controls their dynamics. Three specific aims will be pursued: 1) Construct a map of the outer plate that portrays its native structure before and after microtubule attachment;2) Screen putative outer plate components for location and role in outer plate formation and microtubule attachment;3) Determine the roles of CENP-F and Heel in lateral and end-on attachments to microtubules. The approach is innovative because it combines electron tomography with molecular identification and functional depletions, and will provide the first high-resolution images of the kinetochore outer plate in its native, hydrated form. The research is significant because the structural knowledge it provides will be critical for understanding the location of molecular components in the kinetochore outer plate, rearrangements that occur upon microtubule binding, and the role of outer plate proteins in forming a fibrous network and binding microtubule plus ends. This project is relevant to public health because kinetochore dysfunction has been linked to a number of major health problems including cancer, birth defects, and miscarriages. The increased understanding of kinetochore structure and function will guide efforts for developing pharmacological strategies to control and regulate cell division during the treatment of cancer and other cellular proliferative disorders.